Local structural plasticity of the Staphylococcus aureus evasion protein EapH1 enables engagement with multiple neutrophil serine proteases

Timothy J Herdendorf,Daphne A.C Stapels,Suzan H.M Rooijakkers,Brian V Geisbrecht

doi:10.1074/jbc.ra120.013601

Timothy J Herdendorf, Daphne A.C Stapels + Show 2 more

Open Access

https://doi.org/10.1074/jbc.ra120.013601

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Abstract

Members of the EAP family of Staphylococcus aureus immune evasion proteins potently inhibit the neutrophil serine proteases (NSPs) neutrophil elastase, cathepsin-G, and proteinase-3. Previously, we determined a 1.8 Å resolution crystal structure of the EAP family member EapH1 bound to neutrophil elastase. This structure revealed that EapH1 blocks access to the enzyme's active site by forming a noncovalent complex with this host protease. To determine how EapH1 inhibits other NSPs, we studied here the effects of EapH1 on cathepsin-G. We found that EapH1 inhibits cathepsin-G with a Ki of 9.8 ± 4.7 nm Although this Ki value is ∼466-fold weaker than the Ki for EapH1 inhibition of neutrophil elastase, the time dependence of inhibition was maintained. To define the physical basis for EapH1's inhibition of cathepsin-G, we crystallized EapH1 bound to this protease, solved the structure at 1.6 Å resolution, and refined the model to Rwork and Rfree values of 17.4% and 20.9%, respectively. This structure revealed a protease-binding mode for EapH1 with cathepsin-G that was globally similar to that seen in the previously determined EapH1-neutrophil elastase structure. The nature of the intermolecular interactions formed by EapH1 with cathepsin-G differed considerably from that with neutrophil elastase, however, with far greater contributions from the inhibitor backbone in the cathepsin-G-bound form. Together, these results reveal that EapH1's ability to form high-affinity interactions with multiple NSP targets is due to its remarkable level of local structural plasticity.

Highlights

Members of the extracellular adherence domain proteins (EAPs) family of Staphylococcus aureus immune evasion proteins potently inhibit the neutrophil serine proteases (NSPs) neutrophil elastase, cathepsin-G, and proteinase-3
S. aureus expresses three different EAP domain proteins that act as high-affinity NSP inhibitors (7, 21), a majority of our molecular level understanding on how EAP proteins exhibit this function has come from studies on EapH1 (21, 25, 26)
We found that the Ki value obtained by globally fitting a series of colorimetric assay reaction progress curves (i.e. 9.8 Ϯ 4.7 nM) was in good agreement to that previously reported for a fluorescence-based method (i.e. 4.2 Ϯ 2.2 nM) (21)

Summary

The abbreviations used are: used in this article

MPO, myeloperoxidase; NSP, neutrophil serine protease; NE, neutrophil elastase; CG, cathepsin-G; PR3, proteinase-3; EAP, extracellular adherence protein repeat; Eap, S. aureus extracellular adherence protein; EapH1, S. aureus Eap homolog 1; EapH2, S. aureus Eap homolog 2; RMSD, root-mean-square deviation; SA2PF-pNA, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; PDB, Protein Data Bank. There is heavy selective pressure for such organisms to evolve the capacity to disrupt, inhibit, or otherwise escape from the earliest steps of the innate immune response In this regard, a large body of work has shown that the Gram-positive bacterium Staphylococcus aureus secretes an array of immune evasion proteins that interfere with the central processes of opsonophagocytosis and subsequent killing within the neutrophil phagosome (16 –19). To better understand the physical basis behind NSP inhibition by EAP domain proteins, we solved a crystal structure of EapH1 bound to the most abundant NSP, NE (21) This structure revealed that EapH1 forms a highly complementary but noncovalent complex with NE that blocks substrate access to the protease active site (21).

Results

Discussion

Experimental procedures